Azole antifungal resistance has emerged as a significant problem in the management of infections caused by a number of fungal species including Candida. This problem has had a significant impact in immune-compromised patient populations, particularly those suffering from AIDS. While the use of highly active antiretroviral therapy (HAART) has reduced the frequency of OPC among AIDS patients in the United States, limited access to such therapy in underdeveloped countries, poor compliance, and toxicity associated with HAART will likely contribute to an increase in this problem among AIDS patients world-wide. Understanding the molecular basis of azole resistance will facilitate the development of therapeutic strategies to circumvent this problem and improve the utility of the azole class of antifungal agents. The aim of this proposal is to identify and characterize novel molecular mechanisms of azole antifungal resistance in Candida albicans. The central hypothesis behind this proposal is that genes in addition to CDR1, CDR2, MDR1, ERG11 and PDR16 are required for the stepwise acquisition of azole antifungal resistance in C. albicans. We will use an integrated functional genomic and proteomic approach to characterize and compare the gene expression and proteomic profiles of serial isolates within and between multiple matched sets of azole-susceptible and -resistant isolates of C. albicans. These studies will identify genes and gene products that are associated with the azole resistance phenotype. They will also identify genes and gene products that are coordinately regulated with known resistance mechanisms and hence lend insight into the transcriptional regulation of these mechanisms. We will also screen azole resistant isolates for genes required for this phenotype using an antisense cDNA library. Targeted gene disruption and over-expression will be used to assess the role of candidate resistance genes in this process. These mutants will then be examined for changes in in vitro azole susceptibility. Additional studies will examine the nucleotide sequences of key genes for point mutations that may play a role in this process. These studies will further elucidate the molecular basis for azole antifungal resistance and will identify novel targets for future work towards the development of compounds that will both abrogate resistance and enhance the utility of the azole class of antifungal agents.